As used herein, the term "ligand-receptor assay" refers to an assay for an analyte which may be detected by the formation of a complex between a ligand and a ligand receptor which is capable of specific interaction with that ligand. The ligand may be the analyte itself or a substance which, if detected, can be used to infer the presence of the analyte in a sample. In the context of the present invention, the term "ligand", includes haptens, hormones, antigens, antibodies, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific binding partner therefor, i.e., the ligand receptor of the ligand-receptor assay. In the context of the present invention the term "ligand receptor" includes materials for which there is a specific binding partner, i.e., the ligand of the ligand-receptor assay. Those skilled in the art will appreciate that the analyte of interest is a member of a specific binding pair and may be either a ligand receptor or a ligand depending upon assay design.
Ligand-receptor assays are generally useful for the in vitro determination of the presence and concentration of ligands in body fluids, food products, animal fluids, and environmental samples. For example, the determination of specific hormones, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved the medical diagnosis of the human condition. There is a continuing need for simple, rapid, non-instrumental assays for the qualitative, semi-quantitative, and quantitative determination of such ligands in a sample. In many situations, such assays need to be simple enough to be performed and interpreted by non-technical users. In addition, there has existed an unmet need to determine the presence and concentration of multiple ligands in a single assay. For example, the need exists for a rapid analytical tool to determine, in the emergency rooms of hospitals, the presence of the multiple drugs of abuse.
Ligand-receptor assays rely on the binding of ligands by ligand receptors to determine the concentration of ligands in a sample. Ligand-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize ligand receptors in substantial excess over the concentration of ligand to be determined in the assay. Sandwich assays, in which the ligand is detected by binding to two ligand receptors, one ligand receptor labeled to permit detection and the second ligand receptor frequently bound to a solid phase to facilitate separation from unbound reagents, such as unbound labeled first ligand receptor, are examples of non-competitive assays. Competitive assays generally involve ligand from the sample, a ligand analogue labeled to permit detection, and the competition of these species for a limited number of binding sites provided by the ligand receptor. Those skilled in the art will appreciate that many variations of this basic competitive situation have been previously described and will not be discussed in detail herein except where pertinent to the general objectives of this invention. Examples of ligands which are commonly measured by competitive ligand-receptor assays include haptens, hormones and proteins. Antibodies that can bind these classes of ligands are frequently used in these assays as ligand receptors.
Competitive ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of ligand is determined by its effect on the extent of binding between ligand receptor and labeled ligand analogue. The signal observed is modulated by the extent of this binding and can be related to the amount of ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive immunoassay in which the labeled ligand analogue is a ligand-enzyme conjugate and the ligand receptor is an antibody capable of binding to either the ligand or the ligand analogue. The binding of the antibody to the ligand-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound ligand and ligand-enzyme conjugate for antibody binding sites, as the ligand concentration increases the amount of unbound ligand-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
In general, homogeneous assay systems require both an instrument to read the result and calibration of the observed signal by separate tests with samples containing known concentrations of ligand. The development of homogeneous assays has dominated competitive assay research and has resulted in several commercially available systems. Such systems are not, however, capable of providing results for the determination of multiple ligands in a sample in a single-test format not requiring complex instrumentation.
Heterogeneous, competitive ligand-receptor assays require a separation of bound labeled ligand or receptor from the free labeled ligand or receptor and a measurement of either the bound or the free fraction. Methods for performing such assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009. Such methods, however, are not capable of providing semi-quantitative or quantitative results for the determination of ligands in a sample without using additional tests to calibrate the assay response.
The need for ligand-receptor assays that can be performed without the use of complex instrumentation has led to the development of immunoassays that are simple to perform and result in a response that can be visually interpreted. U.S. Pat. Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232, 4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe devices and methods for ligand-receptor assays that develop colored responses for visual interpretation of the results. While such devices provide simple formats for the visual interpretation of assay results, only the presence or absence of ligand can be determined; semi-quantitative or quantitative determinations using these methods require that separate tests utilizing standards of known concentration be performed to establish the relationship between the observed response and the concentration of ligand.
Employing the techniques described for competitive ligand-receptor assays, the intensity of the resulting color is inversely related to the concentration of ligand in the sample such that assay results that are more intense in color than the reference are interpreted to mean that the sample contained ligand at a lower concentration than that represented by the concentration by the reference. A serious drawback, however, to the widespread utilization of such visually interpreted competitive ligand-receptor assays has been this inverse relationship between intensity of the developed signal and sample ligand concentration. This relationship provides that a sample with a low concentration of ligand will produce a large signal in the assay; and conversely a sample with a high concentration of ligand will produce a small signal in the assay. A further disadvantage of such assays is that if the requirement is for a single test to simultaneously determine multiple ligands each of which must be assigned a semi-quantitative value and each of which has specific individual concentration targets, then individual specific reference zones would have to be provided for each ligand to be determined. Under such circumstances, a test for multiple ligands becomes difficult to produce and complex to interpret.
Another prior art approach, a non-competitive immunochromatographic assay, is described in U.S. Pat. Nos. 4,168,146 and 4,435,504. This assay provides a method for quantitatively determining the presence of a single analyte in a sample in a visually interpreted immunoassay but does not permit the assay of multiple analytes without employing multiple devices. Furthermore, in practice this method is restricted to ligands whose sample concentrations are high relative to ligands that are commonly determined by competitive assay technology. Accordingly, this type of approach is of limited utility. Clearly, there is an unmet need for a ligand-receptor assay capable of determining the presence of a multiplicity of ligands in a sample and concurrently providing individualized semi-quantitative results for each ligand. Furthermore, such an assay should produce such results in a format that is simple enough for an non-technical user to correctly perform and interpret. In addition there is a need for broadly applicable quantitative assay methods that are easily performed and interpreted. The inventive methods of this invention and those described in allowed U.S. patent application Ser. No. 295,560 meet these requirements.
Methods to prepare monoclonal antibodies to ligands which, by themselves, do not generate an immunological response are well known to those skilled in the art. The ligand, or an analogue thereof, is generally coupled, chemically, to a carrier molecule, e.g., a protein, peptide, or other polymer, to form an immunogen (one example of a ligand analogue conjugate as defined herein) which illicits an immunological response. Antibodies are thus raised to the surface of the carrier molecule onto which is coupled the ligand. The selection or screening of antibodies is then performed to choose the antibody which best fulfills the intended use of the antibody. The screening of antibodies is well known to those skilled in the art and is generally performed by binding a ligand-carrier conjugate to a solid phase, allowing the raised antibody to bind to the ligand-carrier conjugate and detecting the presence of the bound antibody with a labelled anti-antibody conjugate. An inherent problem with the generation and screening of antibodies is the difficulty in determining the location of binding of the antibody to the ligand, i.e., the binding site; that is, it is not clear which portion of the ligand analogue is bound by the antibody. This can result in the selection of antibodies which possess a very small but definite affinity to the carrier molecule, or to the chemical structure (herein called the "linkage site") which attaches the ligand analogue to carrier molecule. Such an antibody, will thus bind (an occurrence known as crosstalk) to other, or uncomplementary, carrier molecule-ligand complexes having such a linkage, and produce false positive results when such other complexes are present in a test.
The crosstalk problem has previously been dealt with by using different linkage chemistries for attaching the ligand to the carrier molecule, and for screening the ligand-carrier conjugate; for example, see Van Weemen and Schuurs, The influence of heterologous combinations of antiserum and enzyme-labeled estrogen on the characteristics of estrogen enzyme-immunoassays, Immunochemistry, 12, 667 (1975). In developing ligand receptor assays for a multitude of ligands, however, creating such different chemical structures for multiple ligands can be very time consuming and expensive. In addition, this approach to developing ligand receptors is not guaranteed to be successful because the similarities of linkage chemistries, in general, make all linkage chemistries similar to a certain extent. Also, if one is attempting to prepare ligand receptors with a high degree of specificity for two molecules with very similar structures, one may be limited to the numbers of ligand analogues which can be synthesized.
The development of a ligand receptor assay capable of determining the presence of a multiplicity of ligands requires that the ligand receptors not have a substantial affinity for uncomplementary ligand analogue conjugates. A substantial affinity of a ligand receptor for an uncomplementary ligand analogue conjugate in a multi-analyte assay results in false positive results and renders the assay useless.
The present invention is related to reagents, and method of their use, which reduce or prevent the crosstalk or the undesirable interactions between ligand receptors and uncomplementary ligand analogue conjugates. The reagents, or crosstalk inhibitors, described herein mimic the chemical structure which links the ligand or ligand analogue to the carrier molecule. The extent to which the crosstalk inhibitor must or must not resemble the chemical structure of the ligand analogue linkage chemistry depends on the affinity that the ligand receptor possesses for the linkage chemistry. Thus, the reagents described herein allow the simultaneous determination of the presence of a multiplicity of ligands in a sample by inhibiting or reducing the low affinity interactions of the ligand receptors for the uncomplementary ligand-carrier molecule conjugate or other uncomplementary ligand analogue conjugates.